Mass vaccination would be the simplest and most effective approach to control viral pandemics, such as pandemic and inter-pandemic flu outbreaks, but also to prevent bioterrorist threat, such as the recent terrorist acts involving anthrax in the USA. However, for many viral vaccines such as influenza and smallpox vaccines that are currently produced on egg-based systems, it is most likely that the current production capacity of vaccines manufacturers would not suffice to cover the needs in a case of a pandemics or a bioterrorist attack.
At unpredictable intervals, and in addition to seasonal mild influenza epidemics caused by antigenic drift or reassortment, antigenic shifts with completely new influenza virus subtypes emerge against which immunity in the human population does not exist. They cause global pandemics that spread rapidly around the world. Three of these pandemics occurred in the last century (1918, 1957, 1968). The most severe in 1918, infected approximately 50% of the world's population, of about 25% suffered clinical disease; the total mortality was estimated between 20-40 million, particularly affecting people in the prime of their lives. This pandemic depressed population growth for the following ten years. The last outbreak with high mortality and pandemic potential occurred in 1997, when a new influenza virus (H5N1) emerged in Hong Kong, killing a third of the affected patients, mainly young adults. Fortunately, the virus was not able to spread from person to person and it was possible to quickly stop the outbreak. A similar virus was isolated in 2003 in Hong Kong. In the USA, the impact of the next pandemic is projected to be 18-42 million outpatient visits, 314,000-734,000 hospitalizations and 89,000-207,000 deaths, assuming that the next pandemic will be of a similar magnitude as the 1957 or the 1968 pandemic, and not like the 1918 pandemic (Meltzer M I, Cox N J and Fukuda K. The economic impact of pandemic influenza in the United States: priorities for intervention. Emerging Infectious Diseases 1999; 5:659-671). Extrapolating this projected impact proportionally to the global population, the gross estimate of the global impact of the next pandemic can be estimated at 1-2 billion cases of the flu, 5.3-12.3 million cases of severe illness and 1.5-3.5 million deaths.
Beside a potential pandemic, annual influenza epidemics caused by drifted variants of influenza A and B viruses infect about 10-20% of the population each season, and cause febrile illness, hospitalizations and deaths. Indirect statistical methods have been used to estimate the total burden of influenza; these include various statistical models that quantify the seasonal increase in morbidity and mortality during influenza epidemic periods (Simonsen L., Clarke M J, Williamson G D, Stroup D F, Arden N H, Schonberger L B. The impact of influenza epidemics on mortality: introducing a severity index. Am J. Public Health 1997; 87:1944-1950). Using this methodology, an average influenza season in the USA is currently associated 25-50 million cases of flu, 150,000 hospitalizations, and 20,000-40,000 deaths. Assuming that the age-specific risk of influenza morbidity is similar to that in the USA, the annual average global burden of inter-pandemic influenza may be on the order of around 1 billion cases of flu, around 3-5 million cases of severe illness and 250,000-500,000 deaths (see WHO report, Geneva, April 2003: State of the art of new vaccines Research & Development—Initiative for Vaccine Research).
The currently available influenza vaccines are effective in preventing inter-pandemic influenza-related illness and highly effective in terms of preventing hospitalizations and deaths. In spite of these results, in developed and in developing countries, none of the “high risk population” have been reached as of yet, due in part to the relatively high price of the vaccine and the need for annual re-vaccination. However, very recently, the use of the influenza vaccine has begun to increase worldwide to reach around 235 millions doses in 2003, but there is still a sizable gap in pandemic vaccine demand as the current vaccine production. Indeed the World Health Organization (WHO) estimates that there are about 1.2 billion people at “high risk” for severe influenza outcomes (elderly over 65 years of age, infant, children, adults with chronic health problems, health care workers, . . . ).
The current egg-based system used to produce licensed influenza vaccines, despite being reliable for more than 50 years, shows its limitations that include:                a lengthy, cumbersome and resource-consuming manufacturing process that requires the procurement and quality control of large quantities of eggs for each individual production campaign. This current egg-based production system does not incite additional pharmaceutical companies to go into the business of egg-derived flu vaccines because the potential profit margin is too thin;        the need to select which virus strains will be in the vaccine at least 6 months in advance of the influenza season. This early decision about which strains to include in the influenza vaccine will not always be correct, and the long lead time required to produce the vaccine makes mid-stream corrective action impossible;        the need to produce enough influenza vaccine each year to meet continually increasing demand (about 250 millions doses in industrialized countries in 2004, about 100 millions doses for the USA only). The recent shortfall of influenza vaccines in the USA during winter 2004-2005 due to a contamination in the UK-based plant of an egg-derived flu vaccines manufacturer highlight this issue. Moreover, the current global production capacity of influenza vaccine does not even suffice to cover parts of the global “high risk” population. In reality, it is questionable whether the global infrastructure would be able to handle timely distribution and delivery of pandemic influenza vaccine;        the requirements of hundreds of millions of fertilized chicken eggs to manufacture the vaccine with the associated risks of insufficient supply of eggs in cases of epidemic infections in donor chicken flocks;        the need in cases of life attenuated influenza virus to use costly specific pathogen free (SPF) chicken eggs;        the inflationist costs associated with the use of bovine sera originating from BSE-exempt countries;        the allergenicity of egg-derived components in some individuals;        the inability to use eggs for the propagation of viruses that are highly virulent and lethal to chickens.In addition, current vaccine technology produces vaccines with a narrow spectrum of production, and it is therefore most unlikely that vaccines available in stockpiles would protect against a completely new influenza virus pandemic strain.        
Alternatively, the bioterrorist threat became a major concern for numerous western-countries governments in those past years such as the recent terrorist acts involving anthrax. The United States government takes appropriate measures for rapid diagnosis, defense and reaction to biological attacks through the implementation of the Bioterrorism Preparedness and Response Act in 2002. Biological weapons are indeed relatively accessible and constitute for the bioterrorist organizations a cheap and efficient way to threaten and frighten populations and governments. In particular, the use of the smallpox virus as a biological weapon have increased in recent years and several countries have developed contingency plans to deal with such a risk.
Smallpox is considered as having the greatest potential to cause widespread damage in case of deliberate dissemination, followed by plague, anthrax and botulism. Smallpox was declared eradicated in 1980, and all world countries have since then stopped their vaccination programs. This has led to a steady decline in population immunity to viral infection, which makes of the smallpox virus an even more dangerous agent in case of bioterrorist release. The US Center for Disease Control (CDC) has classified the smallpox as a class A bioterrorist agent, i.e. among the most dangerous micro-organisms given its easy propagation and high mortality rate.
Rapid mass vaccination would be the ideal approach to control an outbreak of smallpox. The US government has taken the lead by securing additional vaccine stock, vaccinating military personnel and key healthcare workers, and establishing a programme for the development of a safe vaccine that could be given to the entire population regardless of their health status. Other governments are monitoring the USA's progress as well as assessing their own emergency preparedness.
In the recent past, governments were either acquiring or producing their own first-generation stocks in state-owned laboratories or issuing commercial tenders. First generation vaccines that were harvested directly from animals, were shown to be effective; however, they were often containing impurities and bacteria that greatly increase the chance of adverse reactions and complications specially in immuno-compromised individuals. Since the eradication of smallpox, a limited number of pharmaceutical companies were able to quickly step in and produce smallpox vaccines. Of that group still fewer are able to produce second-generation vaccines using Dryvax® and Lister-Elstree vaccinia strains in qualified cell cultures according to good manufacturing practice standards. However, as with the first-generation, these vaccines are also not suitable for immune-compromised individuals. A mass vaccination with first and second generation vaccines could lead to complications that would kill one in a million individuals and cause serious disease in 10 times more cases. Consequently, a safer third generation vaccine has been developed by even fewer pharmaceutical companies. Third generation vaccines are based on a strain of the Modified Vaccinia Ankara (MVA) virus used during the smallpox eradication campaign in Germany in the 1970's. In clinical trials, MVA was administered without significant side-effects to about 150,000 individuals, including many considered at risk for the conventional smallpox vaccination.
All these smallpox vaccines are produced on primary chicken embryo fibroblasts isolated from chicken embryos. These production systems are associated with several serious limitations, including:                a lengthy, cumbersome and resource-consuming manufacturing process that requires the procurement and quality control of large quantities of eggs or CEFs for each individual production campaign;        the need in many cases to use costly specific pathogen free (SPF) chicken embryos;        the risks of insufficient supply of eggs in cases of epidemic infections in donor chicken flocks;        the inflationist costs associated with the use of bovine sera originating from BSE-exempt countries;        the allergenicity of eggs in some individuals;        the inability to use eggs for the propagation of viruses that are highly virulent and lethal to chickens.        
While the egg-based and CEFs production process remain relatively reliable process, an efficient cell-based production system would represent a significant improvement in providing a faster, cheaper and less cumbersome method for growing viruses. Moreover, in the event of a flu pandemic, a cell culture based manufacturing process offers additional advantages:                the production of the influenza vaccine can start immediately after the pandemic strain has been identified, isolated and distributed;        there is no need to wait for the development of so-called High Growth Reassortants (viruses adapted to high yield growth in embryonated hens eggs) necessary for production in eggs;        the availability of the first vaccine batch would be approximately 9 weeks after the receipt of the strain, instead of 6-9 months with the egg-derived process;        a cell-derived process allows the production of strains that cannot be adequately grown in eggs (e.g. Avian Hong Kong Flu in 1997);        there is no problem of egg shortage during pandemics.        
Moreover, the use of cell lines for manufacture of viral vaccines, instead of egg or CEF platforms, would have the additional following advantages in connection with the safety of the vaccine: no antibiotic additives present in the vaccine formulation; no toxic preservatives (such as thiomersal) needed; reduced endotoxin levels, no egg allergy issue; growth in protein and serum free media (no adventitious agent/BSE); high purity of virus vaccine preparation.
There is therefore an urgent need to improve on the current viral vaccine production technologies based on eggs or chicken-embryonic fibroblasts. The development of cell-culture platforms as an alternative to the eggs and CEF production systems for the manufacture of viral vaccines is likely the most rapid and promising solution to overcome current vaccine production bottlenecks and time constrains. Moreover, cell-culture production technologies would improve possibilities of up-scaling of vaccine production capacities in face of a pandemic or a terrorist attack.
Based on these specific requirements, the inventor has taken advantage of its expertise in avian biology and in avian embryonic stem (ES) cells to undertake the development of novel stable avian cell lines that enables the efficient replication of human and veterinarian vaccines and vaccine candidates, and that fulfill the industrial, regulatory and medical specifications. Using a proprietary process (see WO 03/076601 and WO 05/007840), the inventor has thus generated a series of well characterized and documented cell lines (the EBx® cells) that are derived from chicken ES cells with no steps of genetic, chemical or viral immortalization. EBx® cells have been generated using a fully documented 2 steps process, and taking in consideration regulatory requirements:
Step 1: Isolation, In Vitro Culture and Expansion of Chicken ES Cells:
Embryonic stem cells are unique in that: (i) they can self-renew indefinitely in vitro as undifferentiated cells, (ii) they have unlimited regenerative capacity, (iii) they maintain a stable chromosomal content; (iv) they express high levels of telomerase and specific cell-surface markers. Despite many efforts worldwide, ES cells have been successfully isolated from only a very limited number of species (mouse, human, monkeys). The inventor has dedicated significant resources over the last years to isolate and establish ES cells from various avian species. Such research efforts led to the successful isolation and characterization of chicken ES cells [Pain et al. 1999. Cell Tissues Organs 165: 212-219]. The inventor then developed proprietary procedures that allow the efficient in vitro culture and large-scale expansion of chicken ES cells without induction of differentiation.Step 2: Derivation of EBx® Cells:Then the inventor established a proprietary process to derive stable adherent and suspension cell lines from chicken ES cells. The process includes the progressive withdrawal of serum, feeder cells and growth factors from the cell culture medium and the adaptation of cells to a suspension culture. These embryonic derived chicken cell lines maintained most of the desirable features of ES cells (ie. indefinite proliferation, expression of ES specific markers such as the telomerase, stability of the karyotype) but in addition displayed new “industrial-friendly” characteristics (growth in suspension in serum-free media).
Based on their attractive biological properties, the inventor selected some chicken EBx® cell lines for further development, such as adherent cell lines EB45 (also named S86N45 in WO 03/076601 and WO 05/007840), from which suspension cell line EB14 has been derived. More preferably the chicken EBx® cells of the invention are selected among EB45 and EB14 cell lines. In a more preferred embodiment, the chicken EBx® cell line is EB14 or its sub-clone EB14-074. For the sake of simplicity, EB14 and EB14-074 will be herein named EB14. EB45 and EB14 cells display an embryonic stem cells phenotype (i.e high nucleo-cytoplasmic ratio) under long term culture (>150 passages). EB45 and EB14 cells are small cells with a large nucleus and nucleolus, and display short pseudopodia extending from the plasma membrane (FIG. 1). EB45 and EB14 cells are highly metabolically active, and present a ribosome and mitochondria rich cytoplasm. A genetic analysis of EB45 and EB14 cells showed that they are male, diploid and genetically stable over the generations (FIG. 2). EB45 and EB14 cells express alcaline phosphatase, stem cells-specific cell surface markers, such as EMEA-1 and SSEA-1 (FIG. 5) and the ES cells-specific ENS1 gene (FIG. 4). Of particular importance EB45 and EB14 cells also express high levels of telomerase enzymatic activity which is stably maintained throughout passages (FIG. 3). Telomerase is a key enzyme in that it promotes continuous cell growth and chromosomal stability. Three weeks and 2.5 months tumorigenicity analysis performed in the immuno-suppressed new-born rat model showed that EB14 cells are non-tumorigenic in vivo. EB45 and EB14 cells are characterized by a very short generation time around 16 hours at 39° C. (the body temperature of chicken) and around 20 h at 37° C. These cell lines present therefore unique properties that make them more efficient, safer and cost-effective cell substrates for the industrial production of viral vaccines such as influenza and smallpox vaccines.
The EBx® cells, and more specifically the EB14 cells of the invention would be of high value for the manufacturing of influenza and smallpox vaccines as well as other major human and animal viral vaccines (Table 1) currently produced on embryonated eggs or on chicken primary fibroblasts, such as the measles, mumps, yellow fever vaccines or investigational poxviruses against infectious diseases such HIV or cancers. Current data have already demonstrated the ability of EBx® cell line, and more specifically to replicate several recombinant and wild-type viruses. For example, preliminary experiments have established that EBx® cells support the replication of influenza virus (see the French priority document patent application FR 05 03583 filed on Apr. 11, 2005, Example 3, pages 30 to 41) and Modified Vaccinia virus Ankara (MVA) (see WO 05/007840).
TABLE 1AVIANSWINEEQUINEHUMANRECOMBINANTinfluenza virusinfluenza virusinfluenza virusSmallpoxCanarypoxreovirusEastern equineinfluenza virusFowlpoxencephalomyelitisfowlpoxWestern equinemeasles virusModified Vaccinia Virus Ankaraencephalomyelitis(MVA)canarypox virusMumps virusAlphavirus - Sinbis viruschicken poxvirusRabiesAlphavirus - Semliki Forest Viruspsittacine herpes virusYellow fever virusAlphavirus - Venezuelan EEVNewcastle Disease Virusthick-bome encephalitisAvian Adenovirus - CELOfalcon herpes viruspigeon herpes virusinfectious bursal disease virusinfectious bronchitis virusMarek's disease virusturkey herpes viruschicken anemia virusavian encephalomyelotis viruspolyomavirus type I & IIAdenovirus type I, II & IIIThe above listed unique properties of EBx® cells, and more specifically EB14 cells, imply the development of a specific process for manufacturing viral vaccines in EBx® cells. Indeed, without to be bound by a theory, the high metabolic level of EBx® cells request that the cell culture medium provide enough energy to cells in order to assure cell growth and viral replication. The aim of the present invention is to provide an innovative and efficient manufacturing process based on the avian embryonic derived stem cells EBx®, more specifically EB14 cells, for the industrial production of a viral vaccines that are currently produced in eggs and in CEFs.